TABLE 2.

Inhibition of purified wild-type hGDH1 and hGDH2 and mutant R443S and R443S/G456A-hGDH1 by female steroidal hormones

The IC₅₀ (± S.E.) values were calculated from the inhibitory curves for each enzyme using the Origin Program. Highly purified enzyme preparations were used for these experiments as described under “Experimental Procedures.” GDH activity was determined in the direction of reductive amination of α-ketoglutarate in TRA buffer, pH 8.0, in the presence of increasing concentrations of female steroidal hormones. DES was varied from 0 to 50 μm, 17β-estradiol and estriol from 0 to 500 μm, and progesterone from 0 to 2000 μm. Estrogen inhibitory curves for the recombinant enzymes were obtained either in the absence of ADP (No ADP) or in the presence of 0.1 and 1.0 mm ADP. The R443S single mutant and the R443S/G456A double mutant were only tested at 0.1 and 1.0 mm ADP, because these enzymes were relatively unstable when assayed without ADP.

	IC50 (μm)
	DES	17β-Estradiol	Estriol	Progesterone
	μm
No ADP
Wild-type hGDH1	1.67 ± 0.06	26.94 ± 1.07	144.77 ± 18.87	118.78 ± 3.63
Wild-type hGDH2	0.08 ± 0.01a	1.53 ± 0.24a	11.34 ± 0.74a	12.31 ± 2.64a
0.1 mm ADP
Wild-type hGDH1	7.06 ± 0.41	69.23 ± 1.31	315.53 ± 26.19	596.39 ± 50.87
R443S-hGDH1	0.49 ± 0.03a	2.22 ± 0.76a	2.27 ± 0.09a	8.61 ± 1.23a
R443S/G456A-hGDH1		4.36 ± 0.36a
Wild-type hGDH2	1.05 ± 0.09a	15.10 ± 1.22a	188.72 ± 17.92b	58.86 ± 24.52a
1 mm ADP
Wild-type hGDH1	26.50 ± 2.24	127.42 ± 10.38	398.39 ± 9.66	743.54 ± 145.98
R443S-hGDH1	2.19 ± 0.14a	14.81 ± 1.05a	42.71 ± 6.98a	78.57 ± 9.19a
R443S/G456A-hGDH1	6.35 ± 0.69a	68.75 ± 7.40a
Wild-type hGDH2	8.38 ± 0.86a	104.40 ± 32.55	274.90 ± 9.19a	392.21 ± 8.92c

^a p < 0.001 compared with the wild-type hGDH1 studied in the presence of the same ADP concentration.

^b p < 0.01 compared with the wild-type hGDH1 studied in the presence of the same ADP concentration.

^c p < 0.05 compared with the wild-type hGDH1 studied in the presence of the same ADP concentration.